Deoxycytidine kinase augments ATM-Mediated DNA repair and contributes to radiation resistance.

Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR).

Enhanced immunoPET of ALCAM-positive colorectal carcinoma using site-specific ⁶⁴Cu-DOTA conjugation.

Activated leukocyte cell adhesion molecule (ALCAM) is an immunoglobulin superfamily cell adhesion molecule that is aberrantly expressed in a wide variety of human tumors, including melanoma, prostate cancer, breast cancer, colorectal carcinoma, bladder cancer and pancreatic adenocarcinoma. This wide spectrum of human malignancies makes ALCAM a prospective pan-cancer immunoPET target to aid in detection and diagnosis in multiple malignancies. In this study, we assess site-specific versus non-site-specific conjugation strategies for (64)Cu-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) immunoPET imaging of a fully human ALCAM cys-diabody (cDb) with a reduced linker length that retains its bivalent binding ability.

Phosphoproteomic profiling reveals IL6-mediated paracrine signaling within the Ewing sarcoma family of tumors.

Unlabelled: Members of the Ewing sarcoma family of tumors (ESFT) contain tumor-associated translocations that give rise to oncogenic transcription factors, most commonly EWS/FLI1. EWS/FLI1 plays a dominant role in tumor progression by modulating the expression of hundreds of target genes. Here, the impact of EWS/FLI1 inhibition, by RNAi-mediated knockdown, on cellular signaling was investigated using mass spectrometry-based phosphoproteomics to quantify global changes in phosphorylation.

Fully automated production of diverse 18F-labeled PET tracers on the ELIXYS multireactor radiosynthesizer without hardware modification.

Unlabelled: Fully automated radiosynthesizers are continuing to be developed to meet the growing need for the reliable production of PET tracers made under current good manufacturing practice guidelines. There is a current trend toward supporting kitlike disposable cassettes that come preconfigured for particular tracers, thus eliminating the need for cleaning protocols between syntheses and enabling quick transitions to synthesizing other tracers. Though ideal for production, these systems are often limited for the development of novel tracers because of pressure, temperature, and chemical compatibility considerations.

Positron emission tomography probe demonstrates a striking concentration of ribose salvage in the liver.

PET is a powerful technique for quantifying and visualizing biochemical pathways in vivo. Here, we develop and validate a novel PET probe, [(18)F]-2-deoxy-2-fluoroarabinose ([(18)F]DFA), for in vivo imaging of ribose salvage. DFA mimics ribose in vivo and accumulates in cells following phosphorylation by ribokinase and further metabolism by transketolase.

Compact microfluidic device for rapid concentration of PET tracers.

HPLC purification and reformulation of positron emission tomography (PET) tracers can lead to significant dilution of the final product, making it difficult to produce a sufficiently high radioactivity concentration for some applications (e.g. small animal imaging, in vitro assays, and labelling of proteins with prosthetic groups).

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.

Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al.

A practical approach to automate randomized design of experiments for ligand-binding assays.

Background: Design of experiments (DOE) is utilized in optimizing ligand-binding assay by modeling factor effects. To reduce the analyst's workload and error inherent with DOE, we propose the integration of automated liquid handlers to perform the randomized designs.

Results: A randomized design created from statistical software was imported into custom macro converting the design into a liquid-handler worklist to automate reagent delivery.

The separation and detection of PET tracers via capillary electrophoresis for chemical identity and purity analysis.

CE coupled with UV detection was assessed as a possible platform for the chemical identity and purity analysis of positron emission tomography (PET) tracers using [(18)F]FAC and [(18)F]FLT as examples. Representative samples containing mixtures of the tracers plus well-known impurities, as well as real radioactive samples (formulated for injection), were analyzed. Using MEKC with SDS in a neutral phosphate buffer, the separation of all compounds in the samples was achieved with baseline resolutions in less than 4.

Quantitative immunoPET of prostate cancer xenografts with 89Zr- and 124I-labeled anti-PSCA A11 minibody.

Unlabelled: Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%-100% of local prostate cancers and 87%-100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using (124)I- and (89)Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer.

Methods: A11 anti-PSCA minibody was alternatively labeled with (124)I- or (89)Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1×PSCA or LAPC-9 xenografts.

Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin.

Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively.

Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo.

The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.

A microreactor with phase-change microvalves for batch chemical synthesis at high temperatures and pressures.

We present a simple microreactor with dimethyl sulfoxide (DMSO) phase-change valves suitable for performing batch organic chemistry under high temperature and pressure conditions. As a proof of principle, we demonstrate a radiofluorination reaction important in the synthesis of [(18)F]FAC, a new positron emission tomography biomarker for immune system monitoring and prediction of chemotherapy response. We achieved high radioactivity recovery (97 ± 1%, n = 3) and conversion efficiency (83 ± 1%, n = 3), comparable to that achieved with macroscale systems, but with a volume 30× smaller.

Metastatic castration-resistant prostate cancer reveals intrapatient similarity and interpatient heterogeneity of therapeutic kinase targets.

In prostate cancer, multiple metastases from the same patient share similar copy number, mutational status, erythroblast transformation specific (ETS) rearrangements, and methylation patterns supporting their clonal origins. Whether actionable targets such as tyrosine kinases are also similarly expressed and activated in anatomically distinct metastatic lesions of the same patient is not known. We evaluated active kinases using phosphotyrosine peptide enrichment and quantitative mass spectrometry to identify druggable targets in metastatic castration-resistant prostate cancer obtained at rapid autopsy.

Inhibition of CSF-1 receptor improves the antitumor efficacy of adoptive cell transfer immunotherapy.

Colony stimulating factor 1 (CSF-1) recruits tumor-infiltrating myeloid cells (TIM) that suppress tumor immunity, including M2 macrophages and myeloid-derived suppressor cells (MDSC). The CSF-1 receptor (CSF-1R) is a tyrosine kinase that is targetable by small molecule inhibitors such as PLX3397. In this study, we used a syngeneic mouse model of BRAF(V600E)-driven melanoma to evaluate the ability of PLX3397 to improve the efficacy of adoptive cell therapy (ACT).

miRNA and mRNA cancer signatures determined by analysis of expression levels in large cohorts of patients.

Toward identifying a cancer-specific gene signature we applied surprisal analysis to the RNAs expression behavior for a large cohort of breast, lung, ovarian, and prostate carcinoma patients. We characterize the cancer phenotypic state as a shared response of a set of mRNA or microRNAs (miRNAs) in cancer patients versus noncancer controls. The resulting signature is robust with respect to individual patient variability and distinguishes with high fidelity between cancer and noncancer patients.

Engineered antibodies for molecular imaging of cancer.

Antibody technology has transformed drug development, providing robust approaches to producing highly targeted and active therapeutics that can routinely be advanced through clinical evaluation and registration. In parallel, there is an emerging need to access similarly targeted agents for diagnostic purposes, including non-invasive imaging in preclinical models and patients. Antibody engineering enables modification of key properties (immunogenicity, valency, biological inertness, pharmacokinetics, clearance route, site-specific conjugation) in order to produce targeting agents optimized for molecular imaging.

Optimization of microfluidic PET tracer synthesis with Cerenkov imaging.

Microfluidic technologies provide an attractive platform for the synthesis of radiolabeled compounds. Visualization of radioisotopes on chip is critical for synthesis optimization and technological development. With Cerenkov imaging, beta particle emitting isotopes can be localized with a sensitive CCD camera.

A semi-automated vascular access system for preclinical models.

Murine models are used extensively in biological and translational research. For many of these studies it is necessary to access the vasculature for the injection of biologically active agents. Among the possible methods for accessing the mouse vasculature, tail vein injections are a routine but critical step for many experimental protocols.

Simplified programming and control of automated radiosynthesizers through unit operations.

Background: Many automated radiosynthesizers for producing positron emission tomography (PET) probes provide a means for the operator to create custom synthesis programs. The programming interfaces are typically designed with the engineer rather than the radiochemist in mind, requiring lengthy programs to be created from sequences of low-level, non-intuitive hardware operations. In some cases, the user is even responsible for adding steps to update the graphical representation of the system.

Analysis of complete genomes of Propionibacterium acnes reveals a novel plasmid and increased pseudogenes in an acne associated strain.

The human skin harbors a diverse community of bacteria, including the Gram-positive, anaerobic bacterium Propionibacterium acnes. P. acnes has historically been linked to the pathogenesis of acne vulgaris, a common skin disease affecting over 80% of all adolescents in the US.

Plug-and-play modules for flexible radiosynthesis.

We present a plug-and-play radiosynthesis platform and accompanying computer software based on modular subunits that can easily and flexibly be configured to implement a diverse range of radiosynthesis protocols. Modules were developed that perform: (i) reagent storage and delivery, (ii) evaporations and sealed reactions, and (iii) cartridge-based purifications. The reaction module incorporates a simple robotic mechanism that removes tubing from the vessel and replaces it with a stopper prior to sealed reactions, enabling the system to withstand high pressures and thus provide tremendous flexibility in choice of solvents and temperatures.

On-demand droplet loading for automated organic chemistry on digital microfluidics.

Organic chemistry applications on digital microfluidic devices often involve reagents that are volatile or sensitive and must be introduced to the chip immediately before use. We present a new technique for automated, on-demand loading of ~1 μL droplets from large (~1 mL), sealed, off-chip reservoirs to a digital microfluidic chip in order to address this challenge. Unlike aqueous liquids which generally are non-wetting to the hydrophobic surface and must be actively drawn into the electrowetting-on-dielectric (EWOD) chip by electrode activation, organic liquids tend to be wetting and can spontaneously flood the chip, and hence require a retracting force for controlled liquid delivery.

NEMA NU-4 performance evaluation of PETbox4, a high sensitivity dedicated PET preclinical tomograph.

PETbox4 is a new, fully tomographic bench top PET scanner dedicated to high sensitivity and high resolution imaging of mice. This manuscript characterizes the performance of the prototype system using the National Electrical Manufacturers Association NU 4-2008 standards, including studies of sensitivity, spatial resolution, energy resolution, scatter fraction, count-rate performance and image quality. The PETbox4 performance is also compared with the performance of PETbox, a previous generation limited angle tomography system.